Scanning spectrophotometer for high throughput fluroescence detection

ABSTRACT

A fluorescence spectrophotometer having an excitation double monochromator, a coaxial excitation/emission light transfer module, and an emission double monochromator. Each monochromator includes a pair of holographic concave gratings mounted to precisely select a desired band of wavelengths from incoming broadband light without using other optical elements, such as mirrors. Selected excitation light is directed into a sample well by a light transfer module that includes a coaxial excitation mirror positioned to direct excitation light directly to the bottom of a well of a multi-well plate. Fluorescence emission light that exits the well opening is collected by a relatively large coaxial emission mirror. The collected emission light is wavelength selected by the emission double monochromator. Selected emission light is detected by a photodetector module.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of the priority of U.S. ProvisionalApplication Serial No. 60/130,438, filed Apr. 21, 1999 and entitled“Novel Scanning Spectrophotometer For High Throughput FluorescenceDetection.”

TECHNICAL FIELD

This invention relates to wavelength scanning fluorescencespectrophotometers using dual grating monochromators, but not opticalfilters, to select excitation and emission wavelengths of light and todetect and quantify simultaneous fluorescence emission from multiplefluorophores in the same sample.

BACKGROUND

Definitions:

1) Fluorescence: The result of a multi-stage process of energyabsorption and release by electrons of certain naturally occurringminerals, polyaromatic hydrocarbons and other heterocycles.

2) Excitation: photons of energy, e=hv_(exc), are supplied by a lightsource and absorbed by an outer electron of a fluorophore, which iselevated from the ground state, S₀, to an excited electronic singletstate, S′₁.

3) Excited State Lifetime: An excited electron remains in the singletstate for a finite period, typically from 1 to 20 nanoseconds, duringwhich the fluorophore undergoes a variety of changes includingconformational changes and alterations in the interaction with solvent.As a result of these changes, the energy of the S′₁ singlet electronpartially dissipates to a relaxed singlet excited state, S₁, from whichfluorescence emission of energy occurs, returning the electron to theground state, S₀.

4) Emission: photons of energy, e=hv_(em), are released from an excitedstate electron, which returns the fluorophore to the ground state. Owingto energy loss during the excited state lifetime, the energy of thesephotons is lower than that of the exciting photons, and the emittedlight is of longer wavelength. The difference in the energy (orwavelengths) is called the Stoke's shift and is an important feature inthe selection of a dye for use as a label or in a probe. The greater theStoke's shift, the more readily low numbers of photons can bedistinguished from background excitation light.

5) Fluorophores: Fluorescent molecules are generally referred to asfluorophores. When a fluorophore is utilized to add color to some othermolecule, the fluorophore is called a fluorescent dye and thecombination is referred to as a fluorescent probe. Fluorescent probesare designed to: 1) localize and help visualize targets within aspecific region of a biological specimen, or, 2) respond to a specificstimulus.

6) Electromagnetic Spectrum: the entire spectrum, considered as acontinuum, of all kinds of electric and magnetic radiation, from gammarays, having a wavelength of 0.001 Angstroms to long waves having awavelength of more than 1,000,000 kilometers and including theultraviolet, visible and infrared spectra.

7) Fluorescence Spectrum: Unless a fluorophore is unstable(photobleaches), excitation and emission is a repetitive process duringthe time that the sample is illuminated. For polyatomic molecules insolution, discrete electronic transitions are replaced by broad energybands called the fluorescence excitation and fluorescence emissionspectra, respectively.

8) Monochromator: A device which admits a wide spectral range ofwavelengths from the electromagnetic spectrum via an entrance aperture,and, by dispersing wavelengths in space, makes available at an exitaperture only a narrow spectral band of prescribed wavelength(s).Optical filters differ from monochromators in that they providewavelength selection through transmittance of selected wavelengthsrather than through spatial dispersion. A second distinguishing featureof a monochromator is that the output wavelength(s), and in many cases,the output spectral bandwidth, may be continuously selectable.Typically, the minimal optical components of a monochromator comprise:

(a) an entrance slit that provides a narrow optical image;

(b) a collimator which ensures that the rays admitted by the slit areparallel;

(c) some component for dispersing the admitted light into spatiallyseparate wavelengths;

(d) a focusing element to re-establish an image of the slit fromselected wavelengths; and,

(e) an exit slit to isolate the desired wavelengths of light.

In a monochromator, wavelength selection is achieved through a drivesystem that systematically pivots the dispersing element about an axisthrough its center. Slits are narrow apertures in a monochromator whichmay have adjustable dimensions. Slits effect selection of the desiredwavelength(s) and their dimensions may be adjustable.

9) Double Monochromator: Two monochromators coupled in series. Thesecond monochromator accepts wavelengths of light selected by the firstand further separates the prescribed wavelengths from undesiredwavelengths.

10) Wavelength Scanning: Continuous change of the prescribed outputwavelength(s) leaving the exit slit of a monochromator. In aspectrophotometer, wavelengths of the electromagnetic spectrum arescanned by the excitation monochromator to identify or prescribe thewavelength(s) at which a fluorophore is excited; wavelength scanning bythe emission monochromator is used to identify and detect thewavelength(s) at which a fluorophore emits fluorescent light. Inautomated fluorescence spectrophotometers, wavelength scanning by theexcitation and emission monochromators may be performed eitherseparately or concurrently (synchronous scanning).

11) Area Scanning: Area scanning is distinct from wavelength scanningand is the collective measurement of local fluorescence intensities in adefined two dimensional space. The result is an image, database or tableof intensities that maps fluorescence intensities at actual locations ina two dimensional sample. At its simplest, area scanning may be aphotograph made with a camera in which all data are collectedconcurrently. Alternatively, the sample may be moved past a detectorwhich measures the fluorescence in defined sub-areas of a sample. Thecollected information creates a matrix which relates fluorescenceintensity with position from which an image, table or graphicalrepresentation of the fluorescence in the original sample can becreated.

12) Fluorescence Detectors

Five elements of fluorescence detection have been established throughlaboratory use of fluorophores during the last two decades:

(a) an excitation source,

(b) a fluorophore,

(c) some type of wavelength discrimination to isolate emission photonsfrom excitation photons,

(d) some type of photosensitive response element that converts emissionphotons into a recordable form, typically an electronic signal or aphotographic image, and,

(e) a light tight enclosure to restrict ambient light.

Fluorescence detectors are primarily of four types, each providingdistinctly different information:

(a) Cameras resolve fluorescence as spatial coordinates in twodimensions by capturing an image: [a] as a photographic image on highlysensitive film, or, [b] as a reconstructed image captured on arrays ofpixels in a charge coupled device (CCD).

(b) Fluorescence microscopes also resolve fluorescence as spatialcoordinates in two or three dimensions. Microscopes collect all of theinformation for an image for a prescribed visual field at the same timewithout any movement of either the sample or the viewing objective. Amicroscope may introduce qualitative estimation of fluorophoreconcentration through use of a camera to capture an image in which casethe measure is a function of exposure time.

(c) Flow cytometers measure fluorescence per biological cell in aflowing liquid, allowing subpopulations within a mixture of cells to beidentified, quantitated and in some cases separated. Flow cytometerscannot be used to create an image of a defined area or performwavelength scanning. The excitation light source is invariably a laserand wavelength discrimination is accomplished through some combinationof tunable dye lasers and filters. Although these instruments may employphotomultiplier tubes to detect a measurable signal, there are no flowcytometers that employ monochromators for wavelength scanning.

(d) Spectrofluorometers (spectrophotometer(s)) typically employ a PMT todetect fluorescence but can measure either: [a] the average currentevoked by fluorescence over time (signal averaging), or, [b] the numberof photons per unit time emitted by a sample (photon counting).

Fluorescence spectrophotometers are analytical instruments in which afluorescent dye or probe can be excited by light at specificwavelengths, and, concurrently, have its emitted light detected andanalyzed to identify, measure and quantitate the concentration of theprobe. For example, a piece of DNA may be chemically attached, orlabeled, with fluorescent dye molecules that, when exposed to light ofprescribed wavelengths, absorb energy through electron transitions froma ground state to an excited state. As indicated above, the excitedmolecules release excess energy via various pathways, includingfluorescence emission. The emitted light may be gathered and analyzed.Alternatively, a molecule of interest may be conjugated to an enzymewhich can convert a specific substrate molecule from a non-fluorescentto a fluorescent product following which the product can be excited anddetected as described above.

The ranges of excitation and emission wavelengths employed in afluorescence spectrophotometer typically are limited to the ultravioletand visible portions of the electromagnetic spectrum. For the purposesof fluorescence detection, useful dyes are those which are excited by,and emit fluorescence at, a few, narrow bands of wavelengths within thenear ultraviolet and visible portions of the electromagnetic spectrum.Desired wavelengths for excitation of a specific fluorescent moleculemay be generated from:

1) a wide band light source by passing the light through a series ofbandpass filters (materials which transmit desired wavelengths of lightand are opaque to others), or cut-on filters (materials which transmitall wavelengths longer or shorter than a prescribed value,

2) a narrow band light source such as a laser, or,

3) an appropriate monochromator.

For a wide band light source, the light to which a fluorescent dye isexposed is typically isolated through bandpass filters to select adesired wavelength from the ultraviolet or visible spectrum for use inexcitation. In monochromator-based instruments, the wavelength of choiceis obtained after light from the source has been dispersed into aspectrum from which the desired wavelength is selected. Whatever thelight source, the fluorescence emission is typically isolated throughbandpass filters, cut-on filters, or emission monochromators to select adesired wavelength for detection by removal of all light of anywavelengths except the prescribed wavelengths. Most fluorescencedetection involves examination of specimens that are in a liquid phase.The liquid can be contained in a glass, plastic or quartz containerwhich can take the form of, for example: an individual cuvette; aflow-through cell or tube; a microscope slide; a cylindrical orrectangular well in a multiwell plate; or silicon microarrays which mayhave many nucleic acids or proteins attached to their surfaces.Alternatively, the liquid can be trapped in a two-dimensionalpolyacrylamide or agarose gel. In each of these cases, light which hasalready passed through the optical filters to select the correctwavelengths for excitation illuminates the sample in the container orgel; concurrently, emitted light is also collected, passed through asecond set of optical filters to isolate the wavelengths of emission,and then detected using a camera, or photosensor.

The optical filters used in fluorescence detectors presentcharacteristics that limit the sensitivity, dynamic range andflexibility of fluorescence detection, including: light absorption whichcauses a loss of efficiency through the system; inherentauto-fluorescence, which produces a high background signal; transmissionof other wavelengths outside the wavelengths of desired bandpass which,in turn limits both sensitivity and dynamic range. Optical filters mustbe designed and manufactured to select for discrete ranges ofwavelengths (“center-width bandpasses”) which limits fluorescencedetection to the use of compounds which are excited and emit atwavelengths appropriate for those filters. Development of a newfluorescent dye with unusual spectral properties may necessitate designof a new excitation/emission filter pair.

To increase efficiency in fluorescence cuvette spectrophotometers aswell as to provide continuous selection of wavelengths, it has beenknown to use grating or prism-based monochromators to disperse incominglight from an excitation source, select a narrow band of excitationwavelengths and, separately, to select an emission wavelength. Gratingscome in many forms but are etched with lines that disperse broadbandlight into its many wavelengths. A monochromator typically includes alight-tight housing with an entrance slit and an exit slit. Light from asource is focused onto the entrance slit. A collimating mirror withinthe housing directs the received beam onto a flat optical grating, whichdisperses the wavelengths of the light onto a second collimating mirrorwhich in turn focuses the now linearly dispersed light onto the exitslit. Light of the desired wavelength is selected by pivoting thegrating to move the linear array of wavelengths past the exit slit,allowing only a relatively narrow band of wavelengths to emerge from themonochromator. The actual range of wavelengths in the selected light isdetermined by the dimensions of the slit. The process of continuousselection of a narrow band of wavelengths from all wavelengths of acontinuous spectrum is referred to as wavelength scanning and the angleof rotation of the dispersing optical grating with respect to theentrance and exit slits correlates with the output wavelength of themonochromator. In order to more precisely select the wavelengths ofexcitation and fluorescence detection, it has been known to use twogratings in each monochromator to enhance wavelength selection for boththe excitation and emission light in a fluorescence spectrophotometer.While the monochromators potentially eliminate the need to use opticalfilters for wavelength selection and free the scientist from thelimitations of filters, their use imposes other limitations oninstrument sensitivity and design. For example, monochromators havingthe configurations described above have the disadvantage of requiring atleast four mirrors and two dispersing elements, along with associatedlight blocking entrance and exit slits. Consequently, such devices havebeen relatively complex and comparatively inefficient compared to filterbased instruments.

Analysis of multiple samples in multi-well plates is a highlyspecialized use of fluorescence spectrophotometers. Typically, theexcitation light is introduced into a well from a slight angle above thewell in order to allow the majority of the fluorescence emission lightfrom the sample within a well to be collected by a lens or mirror.However, as the number of wells per plate is increased (e.g., from 96wells per plate to in excess of 9600 per plate), this side illuminationconfiguration becomes disadvantageous, since most of the incomingexcitation light strikes the side of the well rather than the sample.Since such wells typically have black side walls, much of the excitationlight is lost.

As mentioned above, one method employed to overcome the limitations ofside illumination configurations has been use of an optical fiber toguide the excitation light to an illumination end of the fiber directlypositioned over a well. A second bundle of fibers is employed to collectlight from the well and transmit it to the PMT. In a variation of thisdesign, a bifurcated optical fiber positioned above a microwell has beenused to carry light both into and out of the well. However, opticalfibers typically introduce absorption losses and may also auto-fluoresceat certain wavelengths. Accordingly, such a solution is not particularlyefficient.

Another approach has been to use multi-well plates with transparentbottoms, and exposing a sample within a well to excitation light fromthe bottom while collecting emission light from the open top. While thisapproach has value in some circumstances, light is lost from absorptionas well as from light scattering by the plastic at the well bottom.

Additionally, the transparent plate material may itself auto-fluoresce.In addition, well-to-well optical reproducibility of the well bottommaterial has not been achieved, which has limited the ability tocorrelate measurements on a well-to-well or plate-to-plate basis.Accordingly, such a solution has proven to be less efficient thanilluminating and collecting light from the same side of a sample.

Examples of such prior art using fiber optic light paths include asingle unit fluorescence microtiter plate detector (the “SpectromaxGEMINI”) introduced in 1998 by Molecular Devices, which employs a hybridcombination of single grating monochromators, filters, mirrors andoptical fibers, and the “Fluorolog-3” a modular instrument and the“Skin-Sensor”, a unitized instrument, produced by Instruments SA, bothof which employ bifurcated fiber optic bundles to conduct light from anexcitation monochromator and to collect light from a sample after whichit is transmitted to the excitation monochromator.

It should be noted that microtiter plate applications of fluorescencemonochromators are also limited to microwell plates with 384, 96 orfewer wells; that is, 1536-well microplates as well as “nanoplates”containing 2500 wells, 3500 wells and even 9600 wells cannot be usedwith the current fiber-optic/monochromator based instruments. Detectorsfor such plates typically use lasers and filters combined with confocalmicroscopy.

For the large number of applications involving glass microscope slides,polyacrylamide gels or standard 96-well, 384-well and 1536-wellmicrowell plates, it would be desirable to have a fluorescencespectrophotometer that provides high efficiency, enables high precisioncontinuous excitation and emission wavelength selection, providessignificantly greater dynamic range, eliminates the use of opticalfilters and optical fibers (i.e., light paths do not pass through anyoptical materials other than air), and has a highly efficient structurefor both guiding the excitation light onto a sample and collecting theemission light from the sample in a microtiter well or on a twodimensional surface such as a glass microscope slide, polyacrylamidegel, silicon microarray, or other solid surfaces.

In general, the measurement of fluorescent light intensity, theluminescence, is defined as the number of photons emitted per unit time.Fluorescence emission from atoms or molecules can be used to quantitatethe amount of an emitting substance in a sample. The relationshipbetween fluorescence intensity and analyte concentration is:

F=kQ _(e) P _(o)(1-10^([Ebc]))

where F is the measured fluorescence intensity, k is a geometricinstrumental factor, Qe is the quantum efficiency (photonsemitted/photons absorbed), P_(o) is the probability of excitation whichis a function of the radiant power of the excitation source, ε is thewavelength dependent molar absorptivity coefficient, b is the pathlength and c is the analyte concentration. In previous applications, theabove equation was simplified by expanding the equation in a series anddropping the higher terms to give:

F=kQ _(e) P _(o)(2.303ε*b*c).

In the past, this relationship was acceptable because fluorescenceintensity appeared to be linearly proportional to analyte concentration.The equation fails, however, to provide for true comparison of thefluorescence intensities of different fluorophores because measurementof fluorescence intensity is highly dependent upon k, the geometricinstrumental factor.

Different types of detectors vary in both the time period during which ameasurement is made and the speed at which each can discriminate betweenphotons, characteristics which can be of critical importance whencomparing the luminosity of two fluorophores. Consider two fluorescentdyes that differ only in that the excited state lifetime of one istenfold longer than that of the excited state lifetime of the secondfluorophore (e.g., 1 nanosecond and 10nanoseconds, respectively). Whendetected using photographic film exposed for a defined exposure time,the dye with the shorter lifetime would clearly appear brighter on theexposed film. However, if a detector employing continuous excitationwere used which could not discriminate between photons at a high enoughfrequency, the fluorophore with the shorter excited state lifetime couldactually be emitting far more photons but the detector could erroneouslyindicate that the fluorescence intensity of the two dyes was the same.

Fluorescence is detected in spectrophotometers through generation ofphotocurrent in an appropriate photosensitive device such as aphotomultiplier tube or other photosensing device, both of which arecharacterized by low levels of background or random electronic noise.For this reason, fluorescent emission processes are best characterizedby Poisson statistics and fluorescence can be measured through eitherphoton counting or signal averaging:

Photon counting is a highly sensitive technique for measurement of lowlevels of electromagnetic radiation. In photon counting detection,current produced by a photon hitting the anode of a photomultiplier tubewith sufficient energy to begin an avalanche of electrons is tested by adiscriminator circuit to distinguish between random electronic noise andtrue signal. At such light levels, the discreteness of photons dominatesmeasurement and requires technologies that enable distinguishingelectrical pulses that are photon-induced from dark-current impulsesthat originate in the detector (e.g., a photomultiplier tube) from othercauses.

In previous applications of photon counting, the dynamic range ofdetection was restricted by the ability of the detector to discriminatebetween photons closely spaced in time. Additionally, the signal tonoise ratio in photon counting is also a function of the lightintensity. Assume a steady light flux incident on a photocathodeproducing m photoelectrons per second. During any one second, the lightincident on the photocathode is, on average, m photoelectrons with astandard deviation of m^(½). The signal to noise ratio in suchmeasurements is:

S/N=m/m ^(½) =m ^(½)  (1)

Depending upon their frequency and energy, individual photoelectrons canbe counted with a detector of sufficient gain but the precision of anymeasurement can never be better than the limit imposed by equation (1).In its simplest form, a practical photon counting instrument consists ofa fast amplifier and a discriminator set to a low threshold relative tothe input, typically −2 mV, which has been found empirically tocorrespond to the optimum compromise between susceptibility toelectrical pickup and operating the photomultiplier at excessive gain.

Theoretically less sensitive than photon counting and with greatersensitivity to electronic drift, signal averaging uses photocurrent as adirect measure of the incident light signal. The noise associated withthe photocurrent I_(k), taking the system bandwidth (frequency ofresponse), B, into account, is given by the shot noise formula:

S/N=(I _(k)/2eI _(k) B)^(½)  (2)

where e is the electronic charge. The forms of equations 1 and 2 aresimilar since they refer to the same phenomenon and predict essentiallythe same result. In contrast to photon counting, in which the signal isinherently digitized and its dynamic range limited by the speed of thetimer counter, in equation 2 the signal is taken as a continuousvariable of the photocurrent and it is possible to obtain a much largerdynamic range. In practice, however, the analog-to-digital conversionprocess severely limits the dynamic range owing to the slow responsetimes associated with A/D converters having more than 16-bit resolution.

In general purpose fluorescence detection instruments, the light sourcecan be a quartz halogen lamp, a xenon lamp or similar gas dischargelamp, a photodiode or one of many types of lasers. Typically the sampleis exposed to continuous illumination which maintains a relativelystable percentage of the total number of fluorophores in an excitedstate. In these instruments, the cross-sectional dimension of the samplewhich is illuminated is principally determined by slits. In more complexinstruments, including any using imaged light, confocal optics or pointsource illumination, the exciting light beam is shaped and focused bylenses and mirrors onto a single point and a single focal plane in thesample.

SUMMARY

According to various embodiments of the present invention, a wavelengthand area scanning fluorescence spectrophotometer is provided thatincludes an excitation double monochromator, a coaxialexcitation/emission light transfer module, an emission doublemonochromator a high speed timer-counter circuit board and a precisionx-y-z mounting table for use in positioning a sample relative to thefocal plane of the exciting light. Operations of each are directed andcoordinated through a timer-counter board.

Each monochromator includes a pair of holographic concave gratingsmounted to precisely select a desired band of wavelengths from incomingbroadband light. Selected excitation light is directed into a samplewell or onto a two dimensional surface such as a polyacrylamide gel ormicroscope slide by a light transfer module that includes a coaxialexcitation mirror positioned to direct excitation light directly into awell of a multi-well plate or onto a particular area of a gel,microscope slide or microarray. Emitted light that exits the sample iscollected by a relatively large front-surfaced mirror. The collectedemission light is wavelength selected by the emission doublemonochromator. Both monochromators contain three precision matched slitsthat are positioned to restrict unwanted wavelengths whilesimultaneously creating a “near point” source of the desired wavelengthsfor the succeeding stage of the optical path. Emission light that hasbeen isolated in this way is projected onto the photodetector modulewhich converts the received energy into a digital representation of thefluorescence intensity of the sample.

One embodiment includes a fluorescence spectrophotometer system having alight source; a first double monochromator operating to separate andoutput selected wavelengths of light from the light source as excitationlight; a light transfer module for directing substantially all of theexcitation light directly onto a sample, and for collecting, focusing,and directing fluorescence from the sample as emission light; a seconddouble monochromator operating to separate and output selectedwavelengths of the emission light; and a photodetector and analyzer fordetecting the selected wavelengths of emission light and outputting anindication of such detection.

Another embodiment includes a double monochromator having an entranceslit for accepting light; a first optical grating positioned tointercept and disperse the accepted light from the entrance slit; afirst selection slit positioned to intercept at least part of thedispersed light from the first optical grating and select and pass anarrowed range of wavelengths from such dispersed light; a secondoptical grating positioned to intercept and disperse the passed lightfrom the first selection slit; and a second selection slit positioned tointercept at least part of the dispersed light from the second opticalgrating and select and pass a narrowed range of wavelengths from suchdispersed light.

Yet another embodiment includes a light transfer module having an inputmirror, positioned coaxially with an area to be illuminated, fordirecting incoming light to illuminate the area; and an output mirror,positioned coaxially with the area to be illuminated and in reflectivealignment with the input mirror, for collecting, focusing, and directinglight emitted by the area upon illumination.

Another embodiment includes a photon counting photodetector and highspeed timer-counter board which largely eliminates instrument drift,provides great sensitivity while enabling high frequency discriminationof photons for maximum resolution and quantitation and, concurrently,providing significantly greater dynamic range.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of an embodiment of a fluorescencespectrophotometer system.

FIG. 2A is a schematic diagram of another embodiment of a doublemonochromator.

FIG. 2B is a schematic diagram showing a tension band actuator mechanismfor pivoting the gratings of the double monochromator shown in FIG. 2A.

FIG. 3 is a schematic diagram of one embodiment of the light transfermodule shown in FIG. 1.

FIG. 4 is a schematic diagram of a simplified version of a fluorescencespectrophotometer in accordance with the present invention.

FIG. 5A shows a first alternative dual path embodiment of the invention.

FIG. 5B shows a second alternative dual path embodiment of theinvention.

Like reference numbers and designations in the various drawings indicatelike elements.

DETAILED DESCRIPTION

FIG. 1 is a schematic diagram of an embodiment of a fluorescencespectrophotometer system in accordance with the present invention. Abroadband light source 100 illuminates a mirror 102, which is suitablycurved to focus the light onto a double monochromator 104. Inalternative configurations, a narrow band light source such as aphotodiode or a laser can be substituted for the broadband light sourcebut used with the same monochromator configuration.

Light of the desired wavelength is passed by the double monochromator104 to a light transfer module (LTM) 106. The LTM 106 directs theexcitation light from monochromator 104 onto sample 108, which can be,for example, one well of a microwell plate or one lane of a 1-Dpolyacrylamide gel. Any resulting fluorescence emitted by the sample iscollected by LTM 106 which directs the light to the entrance slit ofemission double monochromator 110. The emission monochromator isadjusted to pass wavelengths from the emission spectrum of thefluorophore(s) in the sample and directs those wavelengths tophotodetector 112, which measures the energy of the emitted light.

Photodetector 112 may be any suitable photosensitive device, includingbut not limited to a photomultiplier tube, a phototransistor, or aphotodiode. The electronic output of photodetector 112 is applied to anautomatic processing unit 114, which generates a signal indicatingdetection of the selected emission which is stored in a numerical formsuitable for further analysis. The automatic processing unit 114 may be,for example, a personal computer having a data collection interface tothe spectrophotometer system. In general, all of the elements of theoptical pathways in this instrument, including double monochromators 104and 110 and the LTM 106, should be isolated in light-tight boxes coatedinternally with non-fluorescent absorptive material to minimizereflectances and light from other sources such a room light.

FIG. 2A is a schematic diagram of an embodiment of a doublemonochromator in accordance with the present invention. The illustratedconfiguration may be used for either the excitation double monochromator104 or the emission double monochromator 110.

Broadband light is introduced through a slit 200 in the doublemonochrormator and reflects off the front surface of a first holographicconcave grating 202 that is pivotable around an axis 203. Use of frontsurface reflection enhances the efficiency of the double monochromatorby avoiding absorption of the light within an optical support structure,such as in a rear-surface reflection glass mirror. Use of a concavegrating allows the light to be dispersed into selectable wavelengthswithout the use of supplemental collimating mirrors. This design makesit possible to eliminate two collimating mirrors per grating used inconventional dual monochromator instruments. Use of a holographicgrating also reduces astigmatic aberrations and thus decreases theamount of light of unwanted wavelengths (“stray light”).

Each double monochromator has three slits: an entrance slit, throughwhich light first enters the monochromator; an internal selection slit;and, an exit selection slit, through which light leaves themonochromator. The first concave grating 202 reflects the wavelengths ofthe incoming light as a first spatially dispersed beam 204. Eachwavelength of this first spatially dispersed beam 204 is reflected at aunique angle relative to other wavelengths. By pivoting the firstconcave grating 202 around its axis 203, a desired band of wavelengthscan be directed through the internal selection slit 206 within themonochromator housing.

The selected range of wavelengths 208 is then reflected off a secondholographic concave grating 210 that is pivotable about an axis 211. Thesecond concave grating 210 reflects the wavelengths of the firstspatially dispersed beam 204 as a second spatially dispersed beam 212.By pivoting the second concave grating 210 around its axis 211, adesired narrow band of wavelengths 214 can be directed through an exitselection slit 216.

The widths of selection slits 206 and 216 determine the selectedwavelengths of light leaving the monochromator. Wider slits allow morelight energy to pass through a monochromator, but the light includes abroad range of wavelengths; narrower slits reduce the amount of lightpassing through a monochromator but narrow the selected range ofwavelengths. The use of wider slits increases the sensitivity ofdetection, which is beneficial in measurements of total fluorescence inan area or volume as would be the case in measurements made inmicrowells. By contrast, the use of narrow slits increases the spatialresolution which is beneficial in discriminating between differentfluorophores at particular locations as is the case with bands separatedin a polyacrylamide gel. That is, the smaller the slit dimensions, thesmaller the area of detection at the sample. In the case of laser lightsources and pinhole slits, the area of excitation at any given moment isa point that corresponds to a particular data pair representingfluorescent intensity and position.

It is a general rule in optical systems that each optical componentreduces the efficiency of light throughput. Advantages of theconfiguration shown in FIG. 2A include, but are not limited to,elimination of collimating mirrors for redirecting light within thedouble monochromator, such as is the case with traditionalmonochromators. In the present embodiment, only two light directingelements (the first and second holographic concave gratings 202, 210)are required, thus improving overall light efficiency.

In the configuration illustrated in FIG. 2A, the first concave grating202 and the second concave grating 210 are pivoted oppositely and intandem by a suitable mechanism. A mechanism according to one embodimentuses a tension band actuator mechanism for pivoting the gratings 202,210, as shown in FIG. 2B. The gratings 202, 210 are mounted coaxiallywith pivot wheels 250, 252, respectively. A lever arm 254 is connectedto pivot wheel 250 at one end and a screw drive mechanism 256 at theother end. Lever arm 254 is moved along threaded rod 258 as the rod isrotated by motor 260.

Lever arm 254 rotates pivot wheel 250 as it travels along rod 258. Pivotwheel 250 is connected to pivot wheel 252 by a tension band 262.According to the present embodiment, tension band 262 is a 0.003 inchstainless steel band. Tension band 262 causes the pivot wheel 252 andgrating 210 to counter-rotate in tandem with pivot wheel 250 and grating202. A spring 264 connected between a housing and pivot wheel 252maintains tension in tension band 262. The position of the grating canbe determined and controlled by a microcontroller connected to pivotwheel sensor 266 and screw drive sensor 268.

FIG. 3 is a schematic diagram of one embodiment of the LTM 106 shown inFIG. 1. Excitation light enters the LTM 106 through an entrance aperturewhereupon it is directed, either with or without one or more mirrors300, to a coaxial excitation mirror 302. The coaxial excitation mirror302 may be flat, or have some curvature to focus or disperse light asdesired. The coaxial excitation mirror 302 is positioned to direct theexcitation light onto the sample 108. More particularly, the coaxialexcitation mirror 302 may be positioned somewhat off-axis with respectto sample 108, but should be positioned so that substantially all of theexcitation light strikes the sample to achieve maximum illumination.

In one application, each well of a multi-well plate can be positionedbeneath the coaxial excitation mirror 302 by X-Y translation of eitherthe LTM 106 or of the multi-well plate. In another application involvingmonochromators equipped with optional microscope optics, differentregions of intact biological cells that have been mounted on glassslides or culture plates can be separately imaged by using opticalelements in the light path and by positioning the sample beneath thecoaxial excitation mirror 302 by X-Y-Z translation of either the LTM 106or of the glass slides or culture plates. Any fluorescence emission fromone or more fluorophores in a sample is collected by a coaxial emissionmirror 304. The coaxial emission mirror 304 must be concave so as tofocus and direct the emission light, either directly or by one or morelight directing mirrors 306, out of an exit port of the LTM 106. In theembodiment shown in FIG. 3, the emission light exits the LTM 106 fromthe same side that the excitation light enters the LTM 106. However,different placements of the entrance and exit ports can be used bysuitable placement of light directing 300, 306.

The coaxial placement of the excitation mirror 302 and the emissionmirror 304 ensures that a high percentage of the excitation light isdirected onto the sample within a well 108, and that a high percentageof the fluorescence light emitted from the well opening is collected foranalysis.

In the preferred embodiment, all of the mirrors within the LTM 106comprise front, or “first” surface mirrors. Such mirrors have areflective material, such as aluminum, coated onto the surface of asubstrate, such as glass or ceramic onto which light is directed. Thecoating serves as the reflective surface, so that light does notpenetrate the substrate, as in an ordinary second surface mirror. Suchfirst surface mirrors are substantially more efficient.

It will be understood by one of ordinary skill in the art that a numberof different reflecting mirrors may be used to direct the light withinthe LTM 106, as needed. However, it is desirable that the number of suchreflecting surfaces be minimized in order to improve efficiency of theLTM 106. In the preferred embodiment, the coaxial excitation mirror 302is an elliptical mirror approximately 6×9 millimeters in dimension,while the coaxial emission mirror 304 is approximately 75 millimeters indiameter. Other dimensions can be used and generally will vary with thedimensions of the overall instrument.

FIG. 4 is a schematic diagram of a simplified version of a fluorescencespectrophotometer in accordance with an embodiment of the presentinvention. In this embodiment, excitation wavelengths are selected froma broadband light source 100 by means of an excitation doublemonochromator 104. The excitation light is directed by a coaxialexcitation mirror 302 to a sample within a well 108. Fluorescenceemissions are collected and focused by a coaxial emissions mirror 304that is in reflective alignment with the coaxial excitation mirror 302.The coaxial emissions mirror 304 directs the collected and focused lightinto an emission double monochromator 110. The emission doublemonochromator 110 selects a desired emission wavelength and directs thatwavelength to a photodetector 112 for counting and analysis. Thisembodiment minimizes the number of reflective surfaces within the LTM106.

One aspect of the embodiment shown in FIG. 4 is that its compactconfiguration allows for use of the instrument as a dual pathspectrophotometer. That is, a sample can be excited from both the openside of a well 108 or from the bottom side of the well.

FIG. 5A shows a first alternative dual path embodiment of the invention.One or both of the light source 100 and the excitation doublemonochromator 104 optionally can be translated from their normalposition to a position below a multi-well plate such that excitationlight impinges upon a bottom-illumination coaxial excitation mirror302′, which directs excitation light through the transparent bottomsubstrate of a well 108. (If the light source is not translated, lightdirecting mirrors may be interposed to direct light through theexcitation double monochromator 104). Fluorescence emissions emanatingfrom the opening of the well 108 are collected by a coaxial emissionmirror 304. When configured for bottom illumination, the topillumination coaxial excitation mirror 302 can be removed from the lightpath in order to maximize the amount of fluorescent light collected bythe emission mirror 304. Alternatively, the top-illumination excitationmirror 302 (not shown in FIG. 5A) can be left in place, as shown in FIG.4. In either case, the bottom-illumination excitation mirror 302′ is indirect alignment (as opposed to reflective alignment) with the emissionsmirror 304.

FIG. 5B shows a second alternative dual path embodiment of theinvention. In this configuration, for bottom illumination, a set of oneor more redirection mirrors 303A, 303B, are interposed into the normallight path from the excitation double monochromator 104 in order tointercept the excitation light. The intercepted excitation light isredirected to impinge upon a bottom-illumination coaxial excitationmirror 302′, which directs the excitation light through the transparentbottom substrate of a well 108. The top illumination coaxial excitationmirror 302 may be left in place, as shown, or moved out of position whenthe redirection mirrors 303A, 303B, are moved into position. Suchmovement may be accomplished, for example, by translational movement ofa carriage on which all four mirrors 302, 303A, 303B, 302′, are mounted,into or out of the page. Again, the bottom-illumination excitationmirror 302′ is in direct alignment (as opposed to reflective alignment)with the emissions mirror 304.

In all of the configurations shown in FIGS. 4, 5A, and 5B, additionalredirection mirrors may be used as desired to suitably guide light todesired locations within the instrument.

According to an embodiment, a timer-counter board that operates at afrequency in excess of 100 MHz is used. The discriminator module canrespond to photons at frequencies as high as 30 MHz. The dynamic rangeof this system ranges from 0 to more than 30×10⁶ photons, therebyeliminating the dynamic range limitations which previously restrictedthe use of photon counting in fluorescence detection. If the observedcurrent from the PMT is greater than a prescribed threshold establishedat the discriminator, a 5 volt electrical pulse having a duration ofapproximately 2 nanoseconds is produced; the timer counter circuitenumerates these pulses as a function of time, that is, establishes adirect quantitation for photons per unit time.

In a spectrophotometer according to one embodiment, a pre-castpolyacrylamide gel containing fluorescently labeled nucleic acids orproteins was placed on a flat plate in the positioning mechanism of thepresent invention directly under the LTM. With the excitation andemission monochromators set at wavelengths suitable for the fluorescentlabels, the gel was moved back and forth under the LTM until all of thegels area had been traversed. At each point of this travel, afluorescent reading was made and stored as a two dimensional arrayrepresenting the fluorescent emission at each point of the gel. Thedistance between the points was adjusted to yield the best response fora given data acquisition time. In the actual experiments, the gel wasalso scanned as “lanes” representing the path of electrophoresis fromthe sample well at the top of the gel to the base of the gel because thefluorophores in an electrophoresis gel are arrayed in a line rather thanas a point. Each such lane was scanned from the sample well to thebottom of the gel to achieve a substantial reduction in overall datacollection time. As a reference for background, a blank lane was scannedand the data subtracted on a point-by-point basis from the correspondingdata for lanes containing fluorophores. The corrected data were thenanalyzed in two ways. In one analysis, an image was constructed of theoriginal gel which was compared to standard laboratory photographs ofthe same gel for evaluation of standard gel parameters such as migrationdistance, separation of molecules and concentration of molecular speciesas determined separately by the digital image and the film. In the otheranalysis, a “densitometry” plot equivalent to those made for gel lanesfrom autoradiography films using flat bed scanning detectors wascreated. From the database relating fluorescence intensity of a lane,the center of each fluorescent band was identified and a cross sectionalgraph of fluorescent intensities as a function of migration distance wasprepared. From the use of gels prepared with different but known amountsof the same fluorescent labeled nucleic acids, a standard curveestablishing lower and upper limits of sensitivity, resolution, andoverall dynamic range for gel detection were determined.

The “square intensity point spread function” and the “long penetrationdepth” properties of one and two photon absorption processes have beenrecognized as important features in future developments in fluorescencedetection. Both are accomplished by focusing a femtosecond short pulselaser onto a focal plane in a sample to be studied for fluorescence. Ina spectrophotometer according to another embodiment, a laser beam froman appropriate laser was substituted for the quartz halogen or xenonlight source used for excitation. The laser beam was used to illuminatethe entrance slit of the excitation monochromator. Additionalmodifications where needed in some cases included, for example, the useof one or two pinhole slits rather than the standard rectangular slits,and the insertion of an objective lens to focus the light after it hadpassed through the monochromator. The fluorescence emission wascollected through the light transfer module as previously and thefluorescence measured as a function of time, or in the cases of imageformation and area scanning, as a function of time and position of thelight transfer module over the sample as described in gel analysis,above. In this configuration, a pinhole exit slit on the excitationmonochromator was used to image light onto a specimen and each point ofthe image used to excite fluorescence. Moving the sample position in anx-y-z fashion, enabled scanning of areas of the sample to create animage or database. For confocal microscopy, two pinhole slits wererequired as described below. For multi-photon applications, only asingle pinhole slit was used as the exit slit of the excitationmonochromator. For two-photon excitation, a microlens array could beused if needed to focus the beam for high transmittance. In general, theconfiguration was epi-fluorescent although in certain polarizationapplications, excitation was from the bottom and collection of lightfrom the top.

In yet another embodiment, the present invention was applied in thecreation of a scanning fluorescence polarization detector. Fluorescentmolecules in solution, when excited with plane-polarized light, willemit light back into a fixed plane (i.e., the light remains polarized)if the molecules remain stationary during the fluorophore's period ofexcitation (excited state lifetime). Molecules in solution, however,tumble and rotate randomly, and if the rotation occurs during theexcited state lifetime and before emission occurs, the planes into whichlight is emitted can be very different from the plane of the light usedfor the original excitation.

The polarization value of a molecule is proportional to its rotationalrelaxation time, which by convention is defined as the time required fora molecule to rotate through an angle of 68.5°. Rotational relaxationtime is related to the solution viscosity (η), absolute temperature (T),molecular volume (V), and the gas constant (R):

Polarization value∝ Rotational Relaxation Time

${{{Polarization}\quad {value}} \propto {{Rotational}\quad {Relaxation}\quad {Time}}} = \frac{3\quad \eta \quad V}{RT}$

If viscosity and temperature are constant, the polarization is directlyrelated to molecular volume (molecular size), which, in general,correlates well with molecular weight. Changes in molecular volumeresult from several causes, including degradation, denaturation,conformational changes, or the binding or dissociation of two molecules.Any of these changes can be detected as a function of changes in thepolarization value of a solution. Specifically, a small fluorescentmolecule which rotates freely in solution during its excited statelifetime can emit light in very different planes from that of theincident light. If that same small fluorophore binds to a largermolecule, the rotational velocity of the small molecule decreases andthe effect is detected as a decrease in the polarization value.Measurement of the effect requires excitation by polarized light whichcan be obtained using a laser or through light selection using apolarizing filter which only transmits light traveling in a singleplane. In one experiment, the light of a defined wavelength obtainedfrom the excitation monochromator of the present invention was furtherrefined by passing the light through a polarizing filter designated the“polarizer”, to obtain monochromatic, plane-polarized light forexcitation (for the present purposes designated “vertically polarizedlight”). Concurrently, the light path for collecting the emitted lightwas similarly modified by introduction of a second polarizing filter,designated the “analyzer”, which could be rotated to positions eithervertical or horizontal to the plane of the exciting light. When afluorescent sample is solution was introduced into the light pathbetween the “polarizer” and the “analyzer”, only those molecules whichwere oriented properly to the vertically polarized plane absorbed light,became excited, and subsequently emitted light. By rotating theanalyzing filter, the amount of emitted light in the vertical andhorizontal planes could be measured and used to assess the extent ofrotation of the small fluorescent molecule in the solution before andafter binding to a larger molecule.

In yet another application, the invention was utilized in confocalmicroscopy, a method for eliminating one of the fundamental difficultiesof fluorescence microscopy, namely the reduction in spatial resolutionat the focal plane of the microscope owing to out-of-focus light. Aspectrophotometer according to another embodiment was used to create anovel confocal microscope from the embodiment essentially as describedunder laser excitation above to focus a light image on whole cell mountsand achieve both multiple and single photon excitation of thefluorescent labels in a sample. Fluorophores in planes out of the focuswere not illuminated and did not fluoresce.

In confocal imaging, apertures were used in both the excitation andemission light paths in order to focus a cone of light through thespecimen and in the emission light path in order to eliminate scatteredand out-of-plane fluorescence. The development of mode locked dye lasershas made simultaneous multiphoton excitation practical because suchlasers are capable of delivering the available excitation energy to afocal spot in very brief pulses and with sufficient energy to achievetwo photon excitation. In multiphoton imaging, the focal spot providedby the laser excites a sufficiently small volume that, when used inconjunction with the light transmission module of this invention, makesit possible to collect all emission light without a second pinhole sliton the emission side. No emission aperture changes were necessary.

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Forexample, the double monochromators of the invention can be used in othertypes of instruments, and the LTM may be used in a filter-basedspectrophotometer or other optical instruments. As a further example,the LTM may be used to direct input light to an area to be illuminatedand efficiently collect, focus, and direct light emitted (e.g., eitherby reflection or by fluorescence) from the illuminated area.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A fluorescence spectrophotometer systemincluding: (a) a light source; (b) a first double monochromatoroperating to separate and output selected wavelengths of light from thelight source as excitation light; (c) a reflection light transfer modulefor directing substantially all of the excitation light directly onto asample, and for collecting, focusing, and directing fluorescence lightemitted from the sample as emission light; (d) a second doublemonochromator operating to separate and output selected wavelengths ofthe emission light; and (e) a photodetector and analyzer for detectingthe selected wavelengths of emission light and outputting an indicationof such detection.
 2. The fluorescence spectrophotometer system of claim1, wherein the first double monochromator or the second doublemonochromator includes: (a) an entrance slit for accepting light; (b) afirst optical grating positioned to intercept and disperse the acceptedlight from the entrance slit; (c) a first selection slit positioned tointercept at least part of the dispersed light from the first opticalgrating and select and pass a narrowed range of wavelengths from suchdispersed light; (d) a second optical grating positioned to interceptand disperse the passed light from the first selection slit; and (e) asecond selection slit positioned to intercept at least part of thedispersed light from the second optical grating and select and pass anarrowed range of wavelengths from such dispersed light.
 3. Thefluorescence spectrophotometer system of claim 2, wherein the firstoptical grating and the second optical grating are both concavegratings.
 4. The fluorescence spectrophotometer system of claim 3,wherein the concave gratings are holographic concave gratings.
 5. Thefluorescence spectrophotometer system of claim 2, wherein the firstoptical grating and the second optical grating pivot about axes ofrotation for selecting a desired range of wavelengths of light as afunction of angle of rotation.
 6. The fluorescence spectrophotometersystem of claim 2, further including a band drive, coupled to each ofthe first optical grating and the second optical grating, for rotatingthe first optical grating and the second optical grating synchronously.7. The fluorescence spectrophotometer system of claim 1, wherein thereflection light transfer module includes: (a) an excitation mirror,positioned substantially coaxial with a well containing the sample, fordirecting excitation light to illuminate the sample; and (b) an emissionmirror, positioned substantially coaxial with the well containing thesample, for collecting, focusing, and directing fluorescence lightemitted by the sample.
 8. The fluorescence spectrophotometer system ofclaim 7, wherein the emission mirror is a spherical mirror.
 9. Thefluorescence spectrophotometer system of claim 7, wherein the excitationand emission mirrors are first-surface mirrors.
 10. The fluorescencespectrophotometer system of claim 7, wherein the excitation mirror ispositioned to direct excitation light into an opening of the well, andthe emission mirror is positioned to collect fluorescence light emittedfrom the opening of the well.
 11. The fluorescence spectrophotometersystem of claim 7, wherein the well has a transparent bottom substrate,and the excitation mirror is positioned to direct excitation light intothe well through the transparent bottom substrate, and the emissionmirror is positioned to collect fluorescence light emitted from a topopening of the well.
 12. The fluorescence spectrophotometer system ofclaim 11, wherein one or both of the light source and first doublemonochromator are moved to direct excitation light directly onto theexcitation mirror.
 13. The fluorescence spectrophotometer system ofclaim 11, wherein one or more light directing mirrors are positioned todirect excitation light from the first double monochromator to theexcitation mirror.
 14. The fluorescence spectrophotometer system ofclaim 1, wherein the photodetector and analyzer counts the number ofphotons of the detected selected wavelengths of emission light.